Mimicking orchids lure bees from afar with exaggerated ultraviolet signals

Abstract Flowers have many traits to appeal to pollinators, including ultraviolet (UV) absorbing markings, which are well‐known for attracting bees at close proximity (e.g., <1 m). While striking UV signals have been thought to attract pollinators also from far away, if these signals impact the plant pollinia removal over distance remains unknown. Here, we report the case of the Australian orchid Diuris brumalis, a nonrewarding species, pollinated by bees via mimicry of the rewarding pea plant Daviesia decurrens. When distant from the pea plant, Diuris was hypothesized to enhance pollinator attraction by exaggeratedly mimicking the floral ultraviolet (UV) reflecting patterns of its model. By experimentally modulating floral UV reflectance with a UV screening solution, we quantified the orchid pollinia removal at a variable distance from the model pea plants. We demonstrate that the deceptive orchid Diuris attracts bee pollinators by emphasizing the visual stimuli, which mimic the floral UV signaling of the rewarding model Daviesia. Moreover, the exaggerated UV reflectance of Diuris flowers impacted pollinators' visitation at an optimal distance from Da. decurrens, and the effect decreased when orchids were too close or too far away from the model. Our findings support the hypothesis that salient UV flower signaling plays a functional role in visual floral mimicry, likely exploiting perceptual gaps in bee neural coding, and mediates the plant pollinia removal at much greater spatial scales than previously expected. The ruse works most effectively at an optimal distance of several meters revealing the importance of salient visual stimuli when mimicry is imperfect.

cies, pollinated by bees via mimicry of the rewarding pea plant Daviesia decurrens.
When distant from the pea plant, Diuris was hypothesized to enhance pollinator attraction by exaggeratedly mimicking the floral ultraviolet (UV) reflecting patterns of its model. By experimentally modulating floral UV reflectance with a UV screening solution, we quantified the orchid pollinia removal at a variable distance from the model pea plants. We demonstrate that the deceptive orchid Diuris attracts bee pollinators by emphasizing the visual stimuli, which mimic the floral UV signaling of the rewarding model Daviesia. Moreover, the exaggerated UV reflectance of Diuris flowers impacted pollinators' visitation at an optimal distance from Da. decurrens, and the effect decreased when orchids were too close or too far away from the model. Our findings support the hypothesis that salient UV flower signaling plays a functional

| INTRODUC TI ON
The art of deception, involving a range of strategies individuals adopt to change the perception and behavior of others, is commonly practiced by many organisms across the animal and plant kingdoms. Mimicry, a form of deception, allows individuals to conceal their identity and avoid recognition by (more or less) closely imitating the behavior or resembling the appearance of their models (Dawkins & Krebs, 1979). One of the most remarkable examples of these deceptive adaptations is the duping of pollinating animals by plant mimics. Among the 32 families of deceptive plants (Renner, 2006), orchids are undoubtedly the master tricksters.
With an estimate of about one-third of all species lacking floral reward to pollinators (Ackerman, 1986a;Dafni, 1984;Jersáková et al., 2006), orchids deceive by luring food-seeking animals by finetuned mimicry (i.e., Batesian floral mimicry) or general resemblance of rewarding flowers (i.e., generalized food deception; Shrestha et al., 2020). Surprisingly, how plants succeed in their deception despite widespread imperfect mimicry remains poorly understood (Roy & Widmer, 1999;Schiestl, 2005;Vereecken & Schiestl, 2008). In animals, the success of imperfect mimicry has been explained by high-salience traits, which overshadow other "less important" traits (Cuthill, 2014;Kazemi et al., 2014) by being highly discriminable from the background (Frieman & Reilly, 2015). Although high-salience of signals such as attention-grabbing colors and visual patterns occur as frequently in animals (Kazemi et al., 2014) as in plants (Jersáková et al., 2012;Peter & Johnson, 2008, their role in explaining imperfect mimicry in plants has received comparatively less attention (Vereecken & Schiestl, 2008). In this study, we examined the role salient ultraviolet (UV) signaling plays in the imperfect floral mimicry of a rewardless orchid that falsely advertises a reward to attract bees when afar from model plants.
Flowering plants and pollinating insects interact through a wide range of sensory modalities, which affect both the pollinator's foraging behavior and the plant's reproductive success (Glover, 2011;Leonard et al., 2011a). Pollinating insects, in particular bees, make their foraging decisions most effectively by combining visual, olfactory, and somatosensory floral signals (Kulahci et al., 2008;Leonard et al., 2011a), yet their innate preference for conspicuous floral displays usually makes color and contrasting visual patterns the primary means by which plants first attract them (Naug & Arathi, 2007;van der Kooi et al., 2019). Bees, the main flower visitors, have phylogenetically conserved trichromatic vision (Briscoe & Chittka, 2001), which can be conveniently modeled with maximum sensitivity UV (approx. 340 nm), Blue (435 nm) and Green (560 nm) photoreceptors (Chittka & Kevan, 2005). Plants produce striking floral markings and patterns by absorbing and reflecting UV light (Briscoe & Chittka, 2001;Dinkel & Lunau, 2001;Lunau et al., 2006Lunau et al., , 2021Papiorek et al., 2016). Interestingly, it is the UV reflectance display rather than the UV pattern (absorbance and reflectance) that increases insect visitation (Johnson & Andersson, 2002;Klomberg et al., 2019). The high chromatic contrast that such UV signals can generate is thought to enhance color salience in an opponent color system Lunau et al., 2006;Papiorek et al., 2016); however, such chromatic contrast is assumed to work only at relatively short distances of about few centimeters (e.g., UV absorbing "floral guides"; Garcia et al., 2021;Giurfa et al., 1996;Horth et al., 2014;Orbán & Plowright, 2014). This is because bees typically only use the long wavelength green input channel of their visual system to enable fast achromatic processing and detection of small target signals (Klomberg et al., 2019), although some psychophysics shows that alternative chromatic channels may in some cases also be important for bee detection and recognition Morawetz et al., 2013;Zhang et al., 1995). That UV reflectance can also attract pollinator insects from further afield has been posited for decades (Burr et al., 1995;Daumer, 1956Daumer, , 1958 Koski & Ashman, 2014) but remains unverified. Salient UV signals against the background may be particularly relevant for increasing long-distance attractiveness in plants that employ flower mimicry (Dyer, 1996), but the question of their effectiveness is not easily testable because of the flower structures that incorporate many color tones together. To obtain experimental access to this question, it is possible to focus on modulating signals in flowers that display salient UV signals. One such plant is the Australian donkey orchid Diuris brumalis whose two outer petals appear yellow to human vision and also strongly reflect UV that would be conspicuous to the visual system of bees (Burr et al., 1995). Diuris brumalis is a food-deceptive species, which secures pollination by role in visual floral mimicry, likely exploiting perceptual gaps in bee neural coding, and mediates the plant pollinia removal at much greater spatial scales than previously expected. The ruse works most effectively at an optimal distance of several meters revealing the importance of salient visual stimuli when mimicry is imperfect.

K E Y W O R D S
bee sensory ecology, ecological interactions, flower attraction, food deception, orchid floral mimicry, pollination success, salient stimuli, ultraviolet reflectance, visual food deception

T A X O N O M Y C L A S S I F I C A T I O N
Functional ecology resembling the co-occurring rewarding pea plant Daviesia decurrens (Scaccabarozzi et al., 2018). The mimicry signals consist of both color reflectance and inner flower shape, as the outer petals diverge from the pea flower shape (Scaccabarozzi et al., 2018). Whilst the mimicry in size and shape is imperfect, the orchid coloration, with the average color loci corresponding to the UV region, is perceptually similar to the pea model in color space; such overlap (<0.06 color hexagon units) makes the two species not readily distinguishable in the eyes of their bee pollinator, Trichocolletes spp. (Hymenoptera: Collectidae; Scaccabarozzi et al., 2018). Food-deceptive orchids are known for gaining their pollination success not only by resembling a specific rewarding model flower (Dyer et al., 2012;Scaccabarozzi et al., 2018;Schaefer & Ruxton, 2009), but also exaggerating their floral signals that advertise the false reward and thus increase pollinator responses (Ackerman, 1986b). Therefore, we hypothesized that the two outer petals of Diuris function as an exaggerated version (for UV reflectance display) of the floral signal display that Trichocolletes bees normally encounter in the rewarding Daviesia peas. We expected that modulating the exaggerated UV signals of Diuris over a spatial scale would affect pollinia removal when orchids are relatively distant from their model food plants because pollinators are more likely to mistake the orchid for the rewarding model when afar. In order to setup the UV modulation experiments on the distance range that is ecologically relevant for the orchid mimicry success, our study firstly describes the function of pollinia removal in orchids according to their distance from the model pea plants.

| Study system
Endemic to Western Australia, the orchid Di. brumalis produces yellow-brown nectarless flowers between July and August and is pollinated via mimicry of rewarding pea plants (Daviesia spp.) by native Trichocolletes (Colletidae) bees (Scaccabarozzi et al., 2018;Scaccabarozzi, Guzzetti, et al., 2020;Houston et. al., in press). Trichocolletes is a genus of solitary bees that are specialist and speed (visits last <2 s) feeder on pea flowers and display a distinctive and identical behavior on both orchids and peas, confirming that it is successfully deceived. The orchid mimics the papilionaceous flower typical of the pea model and while the visible spectrum differs between the mimic and model flower, they are likely to look similar through a bee visual model (Scaccabarozzi et al., 2018). However, the orchid flower diverges from the pea flower structure by exhibiting two prominent outer petals.
We carried out our study in Di. brumalis populations spread along the Darling Range in Western Australia during 2018, 2019, and 2020 (Table S1). In situ studies and experimental settings were preferred as the orchids are protected by national regulation and their withdrawal is only allowed for few biological materials.

| Floral morphology and color properties
To test the hypothesis that the two outer petals of Diuris may function as an exaggerated version of Daviesia floral signals, we firstly determined whether the outer petals were the component of the Diuris flower with the highest UV spectral reflectance so amplifying the UV reflectance of the pea model. We obtained UV measurements for each floral component (n = 6 flowers) for both orchid and pea plants using a Cary 4000 UV-Vis spectrophotometer (Agilent Technologies) and calculating the average spectral reflectance for each floral part.
Secondly, we measured the size of the flower components of the flower (mid-inflorescence flower) in 10 plants of both Diuris and Daviesia ( Figure S1, Data S1). We obtained for both species a UVsalient signal according to the cut value of Australian flowers following Dyer (1996) (Data S1). Flower components' area was estimated as follows: as flowers of Diuris and Daviesia show little concavity or convexity, the areas of the outer and central floral components of Diuris were estimated by approximating the components to the closest geometric figures, the ellipse (orange) and the circle (green), respectively ( Figure S1). Daviesia standard petals' area was approximated to an ellipse, to which was subtracted a secondary minor ellipse circumscribing the wing and keel petals ( Figure S1, Data S1).
To quantify the contrast of the respective flower signals, we used the bee visual parameters according to Chittka and Kevan (2005) and neural coding that enables converting visual signals sensed by each receptor channel into Excitation values between 0 and 1.0. The visual system was adapted to foliage background with a biologically relevant neural resting excitation value of 0.5 and a contrast of zero (Chittka et al., 1994;Spaethe et al., 2001). This model enables the calculation of absolute contrast values ranging from 0 to 0.5 (maximum contrast) for any stimulus that is different from the background as perceived by the visual system of bees (Table 1).
False color photography in "bee view" format was used to reveal the overall color pattern perceived by bees of Diuris and Daviesia flowers (Figure 2a,b; Methods S1,S2). Spectrometer measurements of flower components of Diuris and Daviesia were converted according to the established bee visual model (Chittka, 1992). The location of color loci was calculated from the mean of reflectance for floral parts of Di. brumalis and Da. decurrens ( Figure 2c).

| Model-mimic distance experiment
To test whether Diuris pollination success varies depending on the distance to the model pea plants, in 2019, we first quantified the distance between an individual orchid and all the surrounding pea models within a quadrat of 30 × 30 m centred on a single orchid plant (N = 122 orchids across five populations; Table S1, Figure S2 Average color reflectance measured on flower components in Diuris (iii) and Daviesia (iv) peaks in the UV bands (black arrows). Color reflectance in the UV wavelengths (300-400 nm) varied between 0.5% and 37% in Diuris sepals and petals and 2.5% and 28% in the pea model. The UV reflectance of Diuris outer petals ranged between 18% and 37% (see Data S1 and S2).
TA B L E 1 Average of excitation values (±SD, standard deviation) of bee photoreceptors (UV, blue, green) according to Chittka (1992) and Chittka et al. (1994) and relative corrected values for Diuris and Daviesia flower components as shown in Figure 1, including Diuris outer petals treated by UV filter. Note: Excitation values range between 0 and 1.0 where a value of 0.5 represents no excitation of the sensory neural channel, and so, the absolute maximum excitation contrast is 0.5 for each respective channel. confirm that treated Diuris outer petals did not excite the UV bee photoreceptor as untreated orchid petals and Daviesia petals did, we analyzed the spectral reflectance measurements for the different floral components using the model of bee vision including treated petals (Chittka, 1992; Table 1). False color photography in "bee view"

| Ultraviolet manipulations experiments
format was applied on Diuris flower with treated outer petals to show the overall color pattern (Figure 2b).
The effect of the UV reflectance filter solution (Kinesys) on the number of Trichocolletes bee visits to Diuris orchids was tested using choice experiments (Methods S1; Data S3) to rule out the potential effect of the UV filter solution on attracting or repelling bee pollinators.
In the first field manipulation experiment in 2019, we tested the hypothesis that UV reflectance enhances orchid pollination success In the second field manipulation experiment, in 2020, we tested the hypothesis that by displaying an exaggerated version of Daviesia's F I G U R E 2 Color patterns perceived by bees in treated and untreated Diuris flowers and untreated Daviesia. (a) Diuris flower photographed in UV before (control, C) and after applying the UV filter on the outer petals (UV treated, T). (b) False color photography in "bee view" reveals the overall color pattern perceived by bees in treated (i.e., application of the UV filter solution) and untreated outer petals of Diuris flower and untreated Daviesia flower. The UV filter is effectively a long-pass filter transmitting all wavelengths above 400 nm, free of fragrance, oil, PABA, alcohol, parabens, and preservatives (Kinesys). Importantly, the UV images of treated outer petals show very similar reflectance properties to the background and stem foliage reflectance, confirming that the experimental manipulation knocked out UV signaling with respect to background coloration. (c) Location of color loci was calculated from the mean of reflectance for floral parts of Diuris brumalis (Db), and Daviesia decurrens (Dd). The calculations were made using the Hexagon color model of bee vision (Chittka, 1992).

This model represents the internal perception of flower colors by bee pollinators, and resultant sectors (u [ultraviolet]; ub [ultraviolet-blue]; b [blue] bg [blue-green]; g [green]; ug [ultraviolet-green]) show how bees likely interpret spectral signals].
attractive UV reflectance, Diuris benefits from pollinators that mistake it for the rewarding model from afar. We quantified pollinia removal within 63 orchid groups randomly selected across three large orchid populations (Populations 1, 2, 3; Table S1). Each orchid group

| Contrasting floral displays of models and mimics
The size of the orchid flower is about three times bigger than the pea flower ( Figure 1a). The outer petals proved to be both the largest component of the orchid flower and the area with the highest UV reflectance (Figure 1b; Figure S1, Data S1). The strength of the UV signaling in Diuris had a contrast value of 0.34, which is 26% greater than the UV channel contrast value of 0.27 in Daviesia standard petals ( Table 1). False color photography in "bee view" revealed the similarity of the overall color pattern perceived by bees of Diuris and Daviesia flowers (Figure 2b).
According to the color model, the petals of Diuris and petals of Daviesia are located in the bee-perceived "ug" (UV-green) and "u" (ultraviolet) sectors of the Hexagon color space related to the excitation of bee photoreceptors and subsequent bee neural coding of information ( Figure 2c, Table 1; see Chittka, 1992;Chittka et al., 1994). with the distance between orchid and pea ( Figure S2, Data S4).

| UV manipulations experiments and orchid success in model plants over distance
The UV filter treatment had no attracting or repelling effect on the pollinators (see Methods S1, Data S3) confirming the pollinator visits were independent from the mean used to screen the UV signal (UV screening spray). Treated petals of Di. brumalis are located in the bee-perceived "g" (green) Hexagon sector and according to Scaccabarozzi et al. (2018) did not excite bee UV photoreceptors ( Figure 2c, Table 1). Secondly, the color model corroborated that the excitation of Green receptor, which is known to be important for how bees efficiently find flowers (Giurfa et al., 1996;Skorupski & Chittka, 2010;Garcia et al., 2021), was not affected by UV filter treatment ( Table 1). False color photography in "bee view" confirmed that the UV filter knocked out UV signaling with respect to background coloration (Figure 2b).
In the first field manipulation experiment, we quantified the number of pollinia removed from treated and control Diuris flowers

| DISCUSS ION
Our results establish that Diuris orchids mimic and exaggerate foliage has very low UV reflectance (Chittka et al., 1994;Dyer, 1996;Spaethe et al., 2001;van der Kooi et al., 2019). control orchids [IN-C] inside the Daviesia's patch (Figure 3b). At closer range, within pea patch, bees apparently recognize plants by spotting other visual traits as the shape of Diuris two outer petals. A color trait may become less effective in ensuring successful mimicry when other secondary traits such as size and shape of the flowers can be better discriminated (Gigord et al., 2002;Johnson & Morita, 2006).
Outside the model patch, however, orchids with UV filter treatment [OUT-T] experienced substantially lower pollinia removal than control ones [OUT-C] (Figure 3b), due to a lack of the salient signal, which is associated with the model trait. Thus, the exaggerated UV signal produced by Diuris outer petals only increased the orchid's pollinia removal when the mimic was further away from its models' patch.
Our findings demonstrate that salient floral UV reflectance plays a critical role in ensuring Diuris pollinia removal and explain why the exaggerated UV signal is strategically relevant in floral mimicry when the model is not very close to the mimic. According to previous theories predicting the effectiveness of the mimic's floral stimuli to decline with distance from its model (Duffy & Johnson, 2017;Johnson & Schiestl, 2016), we also found that the number of pollinia removed from the orchid flowers decreased significantly with the distance between orchid and pea ( Figure S2). However, the strength and direction of this effect may vary across different spatial scales, and conclusions about the importance of floral stimuli will depend on the scales at which studies are undertaken. For example, by examining the mimic-model effect at considerably smaller spatial scales than usually investigated (i.e., tens to hundreds of meters) (Duffy & Johnson, 2017;Johnson et al., 2003;Peter & Johnson, 2008), our results suggest that the exaggerated UV reflectance of Diuris outer petals function to enhance pollination at an optimal model-mimic range of ~8 m. Diuris outer petals might promote pollinator deception via bee cognitive misclassification (Dyer et al., 2012;Johnson & Schiestl, 2016), displaying color frequencies below the optimal range of color discrimination in hymenopteran (i.e., 400-500 nm) (Peitsch et al., 1992), especially for free-flying honeybees (Rohde et al., 2013;von Helversen, 1972). However, these findings might be context dependent and be specifically linked to the spatial distribution and abundance of the model species for Diuris; we expect that the optimal model-mimic range may vary when involved model species characterized by different distribution and density.
But why might the observed distance range from model species be optimal? To understand this question, we must delve into both the neurophysiology and physiology of how bee pollinators perceive their world. When a bee receives sweet tasting nectar reward from a rewarding plant like Da. decurrens, this promotes a sustained positive neural response via the ventral unpaired median (VUM) neurons that permit an association between flower and reward with a sustained spiking response of about 15 s (Hammer, 1993;Perry & Barron, 2013), and can enable simple associative learning of color information (Dyer & Chittka, 2004;Giurfa, 2004). It is also known that precise color memory in both bees and humans requires simultaneous viewing conditions that decay in less than a second once a target model is no longer in view (Dyer & Neumeyer, 2005;Uchikawa & Ikeda, 1981); therefore, being close to a model species might allow a bee to identify potential differences that unmask the deception (von Helversen, 1972). Given that bees may fly up to about 7 m in a second Srinivasan & Lehrer, 1985), we hypothesize the 8 m distance we observed for optimal pollinia removal is beyond the theoretical upper limit where precise color vision operates; at such distances, the bee has to recall from memory what it thought was rewarding and tends to prefer a slightly more salient comparative stimulus, an effect related to peak shift discrimination (Leonard et al., 2011b;Lynn et al., 2005;Martínez-Harms et al., 2014). The fast visits of Trichocolletes bees on both model and mimic flowers (Scaccabarozzi et al., 2018), suggest that Diuris benefits from foraging speed behavior that unfavours the accuracy of bee choices (Chittka et al., 2003). Thus, we propose that orchids like Diuris master deception by employing both exaggerated signaling and by exploiting the perceptual gaps in pollinators' visual processing.
Our results also highlight that we gain a very different under- itation between the plant species also disappeared. Therefore, the importance of exaggerated UV reflectance in attracting pollinators from a range of several meters can be missed and/or mistakenly dismissed if not measured at the scale at which it has its strongest ecologically relevant effect. Such a long-range signal might not be suspected considering the typical acuity range of bee-chromatic vision for stationary stimuli within the confined space of a Y-maze (Giurfa et al., 1996). Overall, our results support the hypothesis that the functional role of UV reflectance signaling is contingent on the relative distance between deceptive and rewarding species and their pollinators; the distance described here operates at spatial scales of meters, which are much greater than expected for floral colors.
The terminal position of the outer petals on a long-stemmed plant ( Figure 1a) likely promotes (wind) movement of this exaggerated UV signal that can be even better perceived from afar by foraging bees (Brock et al., 2016;Stojcev et al., 2011) by acting as a "flag signal." Contributing to a range of floral displays aimed at pollinator senses, UV reflectance acts as an important visual cue in many flowering plant species (Johnson & Andersson, 2002;Klomberg et al., 2019). The high UV reflectance of Diuris outer petals enables bees to find these relatively scarce flowers from a distance of meters. Selection may favor deceptive floral displays capable of longer-range UV signaling that help pollinators such as solitary bees to locate flowers in habitats where the distribution of rewarding model flowers is patchy, explaining why relatively large, salient UV signals with high background contrast have evolved in the mimic (Rohde et al., 2013). By revealing that floral salient UV displays are efficiently used by bees not only at the very close ranges already well-documented but also from further afield at an optimal distance, we may explain how plant deception succeeds despite imperfect floral mimicry. These findings invite us to extend our understanding of the adaptive significance of UV reflectance and salient signaling that plants display in a captivating phenomenon such as floral mimicry and more general in nature.

ACK N OWLED G M ENTS
We thank A. Aromatisi for fieldwork assistance and useful discussions on the study design. We acknowledge C. Best for fieldwork assistance, T. Houston for input in the behavioral ecology of native bees, T. Scalzo, P. Chapman, M. Massi, and C. May for technical and laboratory assistance. We acknowledge the anonymous review-